We found that neuroblastoma × glioma hybrid NG108-15 cells accumulated lipofuscin-like autofluorescent materials during neuronal differentiation in culture in a medium containing 1% fetal calf serum, 1 mM dibutyryl cyclic AMP and 1 mM theophylline. The emission maximum of the lipofuscin-like autofluorescent materials was between 500 and 550 nm. Granules positive to acid phosphatase and periodic-acid Schiff were increased, as were the autofluorescent granules in NG108-15 cells. Thiolprotease inhibitors, N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-4-aminobutylamide (E-64) and acetyl-Leu-Leu-Arg (leupeptin), markedly accelerated the accumulation of the lipofuscin-like autofluorescent materials in NG108-15 cells. On the other hand, activities of lysosomal thiolproteases, cathepsin B, C and L, were increased during neuronal differentiation. Protein content in the cells was gradually increased with the neuronal differentiation, and the rise was significantly accelerated when proteolysis was inhibited by E-64. These results suggest that the lipofuscin-like autofluorescent materials contain peptidic substances as a component, and indicate that the increase in hydrolytic activities of thiolproteases during neuronal differentiation is not enough for the hydrolysis of peptidic substrates, resulting in the accumulation of autofluorescent materials in NG108-15 cells.